Project 1
Mechanisms of sylvatic dengue emergence

Project 2
The interaction between arthropod-borne viruses (arboviruses) and the RNA interference (RNAi) response

Project 3
Competition among dengue virus serotypes, ecotypes and strains

Project 4
Novel synergies for antiviral drug design

Research Coordination Network for Infectious Disease Evolution Across Scales

Science Tools in the Classroom: Bioinformatics, Genomics and More!


Project 1:
Mechanisms of sylvatic dengue emergence

Sarawak study collaborators (from left): David Perera, Jane Cardosa, Katherine Bossart, Kathryn Hanley, Nik Vasilakis, Katie Young and Fiona McCrossin (Brenda Benefit, photographer)                      Large View

The four serotypes of mosquito-borne dengue virus, which infect up to 100 million people worldwide each year, are the agents of dengue fever. Dengue virus is unique among arthropod-borne viruses in that humans serve as the reservoir host for the virus. Nonetheless each of the four serotypes initially emerged into humans from an ancestral, sylvatic cycle between non-human primates, and perhaps other reservoir species, and arboreal Aedes mosquitoes. This cycle is still extant in southeast Asia and West Africa.

West Africa: In conjunction with collaborators at the University of Texas, Institut Pasteur Senegal, Johns Hopkins, the Santa Fe Institute and New Mexico State University, we have initiated a field study in Kedougou, Senegal, in order to understand the mechanisms by which dengue virus and other arthropod-borne viruses like chikungunya and yellow fever emerge out of non-human primates and into humans. Our study seeks to define the landscape elements, vector species, and primate species that enable these viruses to be transmitted to humans. Moreover we are surveying local people for seroconversion to dengue to assess the frequency and consequences of sylvatic dengue infection.

Southeast Asia: With collaborators at the University of Texas Medical Branch and Universiti Malaysia Sarawak (UNIMAS), we have recently initiated a study of the diversity of arboviruses and vectors in Sarawak, Malaysian Borneo, to better understand the potential for new arboviruses to emerge in this region.

Funding for these projects has been provided by NIH grant 1R01AI069145-01A2, an AAAS Women’s International Collaboration Research grant, a Tulane National Primate Research Center Pilot Study and an NMSU Interdisciplinary Research grant

Recent publications:

Hanley, K.A., T.M. Monath, S.C. Weaver, S. L. Rossi, R.L. Richman, N. Vasilakis. 2013. Fever versus Fever: the role of host and vector susceptibility and interspecific competition in shaping the current and future distribution of the sylvatic cycles of dengue virus and yellow fever virus.
Infection, Genetics and Evolution 19:292-311.

Hanley, K.A., M. Guerbois, T. Kautz, M. Brown, S.S. Whitehead S.C. Weaver, N. Vasilakis, P. Marx. 2014. Infection dynamics of sylvatic dengue virus serotype 2 in a natural primate host, the African green monkey. American Journal of Tropical Medicine and Hygiene  91:672-6.

Diallo, D. A.A. Sall, C.T. Diagne, O. Faye, O. Faye, Y. Ba, K.A. Hanley, M. Buenemann, S.C. Weaver, M. Diallo. 2014.  Zika virus emergence in mosquitoes in southeastern Senegal, 2011. PLoS One 9(10):e109442

Althouse, B.M., A.P. Durbin, K.A. Hanley, S.B. Halstead, S. C. Weaver, D.A.T. Cummings. 2014. Viral kinetics of primate dengue infection in non-human primates: A systematic review and individual pooled analysis. Virology 452-453: 237-46. PMID: 24606701; PMCID in process. [PDF]

Project 2.
The interaction between arthropod-borne viruses (arboviruses) and the RNA interference (RNAi) response.

In arthropods, the most important defense against infection by arboviruses is RNAi, the targeted destruction of messenger-sense RNA with homology to a double-stranded RNA trigger. In collaboration with the National Center for Genome Resources and with current and former members of Greg Ebel’s lab at Colorado State University, we are characterizing the small RNA response to dengue virus infection in mosquito and mammalian cells in culture and also in different mosquito lines that vary in their susceptibility to dengue virus infection. Additionally, we are investigating how RNAi shapes the evolution of population diversity within hosts (i.e. quasispecies diversity) in West Nile virus and dengue virus.

Larger View
The figure above shows the size distribution of small RNAs that map to the positive-sense (blue) and negative-sense (red) dengue virus genome in two mammalian (HuH-7 and Vero) and one mosquito (U4.4) cell lines five days post-infection with rDENV-4. Each panel represents the combined virus-derived small RNAs from three independent replicates per cell line. In the left-hand column the Y-axis is scaled to a single standard to represent relative number of virus-derived small RNAs in the three cell lines; in the right-hand column variable Y-axis scales are used to allow visualization of the distribution of virus-derived small RNAs in each cell line. (Schirtzinger et al. 2015)".

This research was supported by a grant from the National Center for Research Resources (5P20RR016480-12), the National Institute of General Medical Sciences (8 P20 GM103451-12), and a competitive pilot award grant from the NM-INBRE Sequencing and Bioinformatics Core at NCGR.

Recent publications:

D.E. Brackney, E.E. Schirtzinger, T. Harrison, G.D. Ebel, K.A. Hanley. 2015, in press. Modulation of flavivirus population diversity by RNA interference. Journal of Virology

Schirtzinger, E.E., C.C. Andrade, N. Devitt, T. Ramaraj, J.L. Jacobi, F. Schilkey, K.A. Hanley. 2015. Repertoire of virus-derived small RNAs produced by mosquito and mammalian cells in response to dengue virus infection. Virology 476:54-60

Project 3:
Competition among dengue serotypes, ecotypes and strains

Competition between two strains of a single pathogen can prevent disease emergence if an established human strain excludes invasion of a novel strain from a zoonotic reservoir. Alternatively competition can facilitate resurgence if a highly virulent strain displaces one that is weakly virulent. The four serotypes of mosquito-borne dengue virus emerged independently from a sylvatic, zoonotic cycle into an endemic cycle between humans and peridomestic Aedes. Subsequently the endemic ecotypes have diversified into separate subtypes. With collaborators Bill Messer at Oregon Health and Sciences University, John Xu and Immo Hansen at NMSU, and Faye Schilkey at NCGR, we are investigating the mechanisms and impact of competition between (i) sylvatic and endemic ecotypes of DENV and (ii) between highly and lowly virulent subtypes of endemic DENV serotype 3 (DENV-3) in mosquito cells in culture and in vivo.

Aedes aegypti

Funding for this study is provided by NIH 1R15AI113628-01

Recent publications:
Pepin, K.M. and K.A. Hanley. 2008. Density-dependent competitive suppression of sylvatic dengue virus by endemic dengue virus in cultured mosquito cells. Vector Borne and Zoonotic Diseases 8:821-8.

Hanley, K.A., J.T. Nelson, E. E. Schirtzinger, S.S. Whitehead, C.T. Hanson. 2008. Superior infectivity for mosquito vectors contributes to competitive displacement among strains of dengue virus. Biomed Central Ecology 8:1.

Pepin, K.M., K. Lambeth, K.A. Hanley. 2008. Asymmetric competitive suppression between strains of dengue virus. Biomed Central Microbiology 8:28.

Project 4:
Novel synergies for antiviral drug design
The genus Flavivirus, comprising 80 species of single-stranded, positive-sense RNA viruses, includes a large number of globally significant emerging pathogens. In the last 50 years many flaviviruses, such as dengue, West Nile, and tick-borne encephalitis viruses, have exhibited dramatic increases in incidence, disease severity and/or geographic range. For example the annual number of cases of dengue hemorrhagic fever cases worldwide has risen from nearly 0 in the 1950’s to 500,000 today. These trends are exacerbated by failure of vector control to limit virus spread as well as the absence of vaccines for viruses such as dengue and West Nile. Thus effective antiviral therapies are urgently needed to ameliorate the disease burden imposed by flaviviruses.  At present, however, no licensed therapies are available for any flavivirus, largely because existing broad-spectrum drugs have failed to show efficacy against flaviviruses or have generated unacceptably high levels of toxicity.

 Large View
To overcome these limitations, it will be necessary to develop not only new antiviral drugs but also innovative strategies to lower their effective dose and thereby mitigate toxicity. The recent discovery that certain FDA-approved fluoroquinolone antibiotics enhance the activity of the RNA interference pathway suggests one promising new strategy. RNA interference is a ubiquitious antiviral defense of eukaryotes that acts by targeting small interfering RNA’s (siRNA’s) to complementary regions in the viral genome; genomes bound to siRNA’s are marked for cleavage and degradation. Because binding of siRNA’s to a target sequence requires nearly perfect complementarity, RNAi imposes selection pressure for viral mutation that disrupts such complementarity. The proposed research will test the hypothesis that enhancing RNAi will amplify the effect of mutagenic nucleoside analogs on dengue virus mutation rate and replication, and that the synergistic effect of RNAi enhancement will accelerate lethal mutagenesis driven by nucleoside analogs. If this hypothesis is correct, the finding of this study will represent a significant advance in the development of new therapies for flaviviral disease.